RNA profiling of non-cultured fibroblasts isolated from pubertal mouse mammary gland sections

Abstract

The epithelium of the pubertal mouse mammary gland grows and invades the mammary fat pad to form a primary ductal network. This outgrowth is tightly controlled by epithelial and stromal factors that are present in the environment around the terminal end buds (TEB) at the growth front and the newly formed ducts. Identifying the contribution that each cell type makes to this regulation is a major challenge. To identify the role that fibroblasts play during this process we have optimised a fibroblast isolation procedure, followed by cell cleanup, RNA extraction, and amplification from non-cultured, freshly isolated fibroblasts from around the TEB as well as the subtending ducts. This was facilitated by the use of mice that constitutively expressed EGFP, which allowed the visualization of the growth front of the pubertal mammary tree under UV light. The isolated RNA is of sufficiently high quality, giving reproducible qRT-PCR results, for transcriptome analysis after RNA amplification.